Structure-function relationships involved in assembly and activation of NADPH oxidase (respiratory burst) have been explored both in vitro and in transfected cell models. This enzyme is an important host defense system of phagocytes responsible for production of superoxide anion and related microbicidal oxidants; oxidase deficiencies cause chronic granulomatous disease. Recent work has also focused on the oxidase as a model for protein-protein interactions relevant in diverse intra- cellular signal transduction cascades. We have established a model in which multiple interactions of conserved Src Homology 3 (SH3) domains in two cytosolic components (p47-phox and p67-phox) with proline-rich target sequences in other oxidase components mediate NADPH oxidase assembly. Both constitutive and regulated SH3 interactions have been elucidated, based on translocation and activation phenomena observed in whole cells. A tail-tail SH3 interaction between p67-phox and p47-phox affects stability of p67-phox in transfected cells, while two other p47-phox SH3 interactions were evident during oxidase activation when the functions of truncated forms of these proteins were compared. Structural determinants for SH3 domain recognition have also been investigated by gene transfer and mutagenesis; binding epitopes were compared with other well characterized SH3-peptide ligand complexes. We also initiated screening of biased peptide libraries to broaden our understanding of binding specificities of all SH3 domains of this system. We have engineered nine phosphorylation site mutations in p47-phox to explore the role of phosphorylation in regulation of SH3 function during oxidase activation. In related work, we have cloned a cDNA encoding a third cytosolic oxidase component with an SH3 motif (p40-phox), which appears to form a high molecular weight complex with the other cytosolic components. We have expressed p40-phox cDNA in a recombinant baculovirus and have affinity purified this protein through interaction with the C- terminus of p47-phox. We have also expressed most oxidase proteins in the yeast two-hybrid system and have demonstrated interactions in these vectors between p40-phox and the N-terminus of p67-phox and between p67- phox and p21-rac, a Ras-related regulator of the oxidase. These studies may be used as a structural basis for designing drugs that can inhibit this important aspect of inflammation or provide insights on other diverse signal transduction systems involving structurally related proteins.